Modified Carba NP test for the detection of carbapenemase production in gram-negative rods: optimized handling of multiple samples

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brazilian journal of microbiology48(2017)242–245

h ttp : / / w w w . b j m i c r o b i o l . c o m . b r /

Medical

Microbiology

Modiﬁed

Carba

NP

test

for

the

detection

of

carbapenemase

production

in

gram-negative

rods:

optimized

handling

of

multiple

samples

Eloiza

H. Campana

∗,1

_,

_Stephanie

_G.

_Chuster

1

_,

_Isadora

_R.

_da

_Silva,

_Raphael

_P.

_Paschoal,

Raquel

R. Bonelli,

Beatriz

M. Moreira,

Renata

C. Picão

UniversidadeFederaldoRiodeJaneiro,InstitutodeMicrobiologiaPaulodeGóes,LaboratóriodeInvestigac¸ãoemMicrobiologiaMédica, RiodeJaneiro,Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Availableonline7December2016

AssociateEditor:AnaLuciaDarini

Keywords:

Carbapenemases Diagnostics

Gram-negativebacteria

a

b

s

t

r

a

c

t

ThemodiﬁedCarbaNPtestpresentedheremaybeavaluabletoolforlaboratories

inter-estedininvestigatingalargenumberofcarbapenemase-producingbacteriainaless-costly

way.Thetestwasevaluated against48carbapenemase-producingand

carbapenemase-non-producing gram-negative bacteria. No false–positive results were obtained, but

false-negativeresultswereobservedwithOXA-23-andGES-carbapenemase-producing

iso-lates.Aeromonassp.arenottestablebyModiﬁedCarbaNP.

anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

licenses/by-nc-nd/4.0/).

Thespread of carbapenemase-producingisolates in

hospi-talsettingsisamajorpublichealthconcern.Earlydetection

ofcarbapenemaseproducersisessentialtoassureadequate

therapyandfavorableoutcomes.1–5_Carba_NP_test_emerged_as

ausefulalternative todetect carbapenemaseproductionin

Enterobacteriaceae,Pseudomonasspp.andAcinetobacterspp.,2,6,7

asrecommended bythe Clinical and LaboratoryStandards

Institute (CLSI).8 _The_test _is_based _on _{acidiﬁcation} _of

phe-nolredwhenimipenemishydrolyzed,evidencedbythecolor

change of the test solution from red to yellow. Carba NP

test advantages over a number of other phenotypic tests

∗ _{Corresponding}_author_at:_Instituto_de_{Microbiologia,}_Universidade_Federal_do_Rio_de_Janeiro,_Cidade_{Universitária,}_Av._Carlos_Chagas

Filho,373–CentrodeCiênciasdaSaúde,BlocoI,RiodeJaneiro,RJ21941-902,Brazil.

E-mail:[email protected](E.H.Campana).

1 _These_authors_contributed_equally_to_this_work.

include speed inprovidingresults, simplicity ofexecution,

objectivenessininterpretationandincreasedsensitivityand

speciﬁcity.1,2,9,10_On_the_other_hand,_sample_processing_may

becomeexpensiveandtimeconsumingifalargenumberof

isolatesare tested.Hereweproposemodiﬁcations tomake

thetestfasterandlessexpensive.Modiﬁcationsincludedthe

omissionofthecentrifugationstep,celldisruptionusingbath

sonication and the use of imipenem/cilastatinas the

sub-strate.

We studied 48 isolates, including negative controls

and Enterobacteriaceae, Pseudomonas aeruginosa, Acinetobacter

http://dx.doi.org/10.1016/j.bjm.2016.09.015

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Table1–Isolatestested,modiﬁedCarbaNPresultsandproteinconcentration.

Isolates Speciesa _{Beta-lactamase}

(Amberclass)b ModiﬁedCarba NPtestresult Protein concentration (␮g/mL±standard deviation)c Highest dilution yielding positiveresult Carbapenemase producers

1 Klebsiellapneumoniae KPC-2(A) Positive <20±5.48 1:128

2 Kluyverasp. KPC-2(A) Positive <20±2.04 1:128

3 Citrobactersp. KPC-2(A) Positive <20±2.31 1:256

4 Enterobactercloacae KPC-2(A) Positive 107.39±2.45 1:16

5 Enterobactercloacae KPC-2(A) Positive 35.73±21.13 1:64

6 Klebsiellapneumoniae KPC-2(A) Positive <20±12.14 1:64

7 Aeromonassp. KPC-2(A) Inconclusived _– _–

8 Aeromonassp. KPC-like(A) Inconclusive – –

13 Enterobacteraerogenes KPC-2(A) Positive 24.42±3.8 1:8

14 Pseudomonasaeruginosa SPM-1(B) Positive 138.09±4.36 1:32

15 Pseudomonasaeruginosa SPM-1(B) Positive 78.81±4.81 1:64

16 Pseudomonasaeruginosa VIM-1(B) Positive 397.27±24.29 1:8

17 Acinetobacterbaumannii SIM-1(B) Positive <20±0.45 1:32

18 Pseudomonasaeruginosa GIM-1(B) Positive 63.3±2.61 1:128

19 Klebsiellapneumoniae NDM-1(B) Positive <20±1.15 1:512

20 Klebsiellapneumoniae IMP-1(B) Positive <20±7.92 1:512

21 Pseudomonasaeruginosa IMP-18(B) Positive 177.15±19.05 1:64

22 Acinetobacterbaumannii OXA-24(D) Positive 570.85±20.56 Undiluted

23 Klebsiellaoxytoca OXA-48(D) Positive 583.22±16.49 Undiluted

24 Acinetobactersp. OXA-58(D) Positive 1045.91±76.27 Undiluted

25 Acinetobacterbaumannii OXA-143(D) Positive 452.67±26.04 Undiluted

26 Acinetobacterbaumannii OXA-23(D)e _Negative _– _–

27 Acinetobacterbaumannii OXA-23(D)f _Negative _– _–

28 Enterobactercloacae GES-5(A) Negative – –

30 Klebsiellapeumoniae GES-16(A) Negative – –

Carbapenemase

non-producers

C1 Pseudomonasaeruginosa GES-1(A) Negative – –

C2 Klebsiellapneumoniae CTX-M-2(A) Negative – –

C3 Pseudomonasaeruginosa OXA-18(D) Negative – –

C4 Escherichiacoli DHA-1(C) Negative – –

C5 Escherichiacoli CTX-M-15(A) Negative – –

C6 Escherichiacoli CTX-M-8(A) Negative – –

C7 Escherichiacoli FOX-5(C) Negative – –

C8 Escherichiacoli MIR-1(C) Negative – –

C9 Escherichiacoli TEM-1(A) Negative – –

C10 Aeromonassp. GES-like(A) Inconclusive – –

C11 Aeromonassp. GES-like(A) Inconclusive – –

C12 Aeromonassp. None Inconclusive – –

C15 PseudomonasaeruginosaATCC None Negative – –

C16 EscherichiacoliJ53 None Negative – –

a _{Identiﬁcation}_determined_by_MALDI-TOF_MS_(Bruker,_Germany). b _{␤-Lactamases}_production_was_assessed_by_PCR_and_sequencing_assays. c _At_the_least_diluted_sample_showing_positive_result.

d _{Inconclusive:}_{red-to-yellow}_color_change_was_observed_in_both_imipenem₋_and₊_solutions. e _This_isolate_show_ISAba-1_positioned_upstream_and_in_the_opposite_{transcriptional}_direction_of_bla

OXA-23. f _This_isolate_does_not_show_ISAba-1_positioned_upstream_bla

OXA-23.

baumanniiandAeromonasspp.producingclassA,BorD

car-bapenemases(Table1).Bacterialstrainswerecultivatedonto

Trypticase soy agar (TSA) (Difco Laboratories) at 37◦C for

18–24h.A10␮Lcalibratedloopfulloftheteststrainwas

inoc-ulatedinto500␮LTris–HCl(20mM–pH7.5)(Invitrogen).The

suspensionwassubjectedtovortexhomogenizationandbath

sonication for 30min (BRANSONIC ULTRASONIC CLEANER,

47kHz±6%,60W)andpreservedonice.Then,30␮Lofthecell

extractwasmixedwith100␮Lofphenolred(Isofar)

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244

containing0.1mMZnSO4 and6mg/mLimipenem/cilastatin

(Merck)(imipenem+).Themixtureswereincubatedat37◦C

for2h.Testswereperformedinduplicateforallisolates.The

colorchangeoftheimipenem-containingvialfromredto

yel-lowororangeindicatedapositiveresult.Threeindependent

observersrecordedtheresultswithnodiscordantreadings.

Toassesstheinferiorlimitofcarbapenemasedetectionby

themodiﬁedCarbaNPproposed,thetestwasalsoperformed

usingdilutedcrudeextracts.Thelastdilutionyieldingpositive

resultwascentrifugedandthesupernatantwassubjectedto

totalproteinquantiﬁcation,whichwasperformedintriplicate

usingthePierceTMBCAProteinAssayKit(ThermoScientiﬁc)

followingthemanufacturer’srecommendations.

Sensibility,speciﬁcity,positiveandnegativepredictive

val-ues (SN, SP, PPV and NPV, respectively) were calculated,

excluding Aeromonas spp. PCR results for carbapenamases

wereconsideredthegoldstandard.SN,SP,PPV,andNPVwere

calculated with the formulasa/(a+c), d/(b+d), a/(a+b) and

d/(c+d),respectively.

Most carbapenemase-producing isolates showed the

expected positive result, indicating that the modiﬁcation

in the extraction protocol did not jeopardize the

sensitiv-ityofthetest (Table1).Proteinconcentrations atthe most

dilutedextractshowingcarbapenemaseactivityvariedfrom

<20␮g/mLto1045.92␮g/mL(Table1).

AllKPCproducingisolatesyieldedpositiveresultsexcept

forAeromonassp.(Table1)whichshowedinconclusiveresults

inrepeatedtestsirrespectiveofthebeta-lactamaseproduced,

asthered-to-yellowcolorchangewasobservedinsolutions

imipenem− and + (Fig. 1).Thisﬁnding was relatedtothe

acidicnatureofthecrudeextractassessedinrepeatedassays

(pH=5.5).AlthoughAeromonas spp.producing acquired

car-bapenemasesarenotcommoncausesofmultidrug-resistant

infections,microbiologistsshouldbeawarethatCarbaNPtest

mightnotbesuitabletoinvestigatecarbapenemase

produc-tioninthesebacteria.Allmetalo-␤-lactamase(MBL)producers

showed positive results at the modiﬁed Carba NP test, as

expected. Of notice, MBL-producing P. aeruginosa required

increasedprotein levelsin crude extracts togenerate

pos-itive results, likely due to difﬁculty in disrupting the cell

wallofsuchisolates.Inagreementwithpreviouswork,2,11,12

allGES-likecarbapenemaseproducingisolatesshowed

nega-tiveresults.MostclassDcarbapenemaseproducingisolates

showed positive results in Modiﬁed Carba NP test, except

thoseproducingOXA-23(Table1).Dilutedcellextracts,

how-ever,showed negativeresults,which isconsistentwiththe

decreasedimipenemcatalyticactivity presentedby

oxacili-nasescomparedtoothercarbapenemases.Isolatescarrying

blaOXA-23showednegativeresults,regardlessofthepresence

or absenceof ISAba1upstream this gene. TheSN,SP,PPV,

andNPVforCarbaNPmodiﬁedwere73.1,100,100and61.1%,

respectively.Positiveresultswereobservedatdifferenttimes

fordifferentcarbapenemases (rangingfrom5minforNDM

andKPCto2hforOXAtype).

Noteworthy,theModiﬁedCarbaNPtestgave

indistinguish-ableresultswhenperformedusingcellextractsobtainedby

probe sonication (data not shown). Despite the fact that

the equipment for bath sonication is cheaper than the

probe-based, it also enables processing a large number of

isolates concomitantly and avoids excessive manipulation

Fig.1–RepresentativeresultsofthemodiﬁedCarbaNP test.Non-carbapenemaseproducers(A,B,HandK), carbapenemaseproducers(C,F,G,I,J,L,M,andO),and

Aeromonasspp.isolates(D,E,K,NandP)withnegative

controlsolutions(−)andtestsolution(+).(A)E.coliJ-53;(B)

P.aeruginosaATCC25922;(C)KPC-2-producingE.cloacae;(D)

KPC-like-producingAeromonassp.(E)KPC-like-producing

Aeromonassp.(F)NDM-1-producingK.pneumoniae;(G)

OXA-48-producingK.oxytoca;(H)CTX-M15-producing

E.coli;(I)OXA-23-producingA.baumannii;(J)

OXA-143-producingA.baumannii;(K)Aeromonassp.(L) IMP-18-producingP.aeruginosa;(M)SPM-1-producingP.

aeruginosa;(N)KPC-like-producingAeromonassp.(O)

GES-16-producingE.cloacae;(P)GES-like-producing

Aeromonassp.

of potential carbapenemase producers in high inoculums,

protectingagainstenvironmentcontamination.

Although other studies have made differentchanges in

CarbaNP,13–15_the_Modiﬁed_Carba_NP_test_presented_here_may

beavaluabletoolforlaboratoriesinterestedininvestigating

several carbapenemase-producing bacteria with decreased

cost.Althoughthesemodiﬁcationsinvolvetheacquisitionofa

sonicationapparatus,itsinitialcostiscounterbalancedbythe

abilitytoprocessseveralisolatesconcomitantlyandbythe

eliminationofthelysisbuffer,whichisespeciallyattractive

forlaboratoriesthatmustimportthisexpensivereagent.We

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pharmaciesmayserveasthesubstratefortheModiﬁedCarba

NPtest, representinganoff-labeluse ofthis medication in

institutionswherethispracticeisallowed.

Conﬂicts

of

interest

Theauthorsdeclarenoconﬂictsofinterest.

Acknowledgments

We are grateful to Ana Cristina Gales, Laurent Poirel and

MariseDutraAsensiforprovidingpositivecontrolsemployed

inthisstudy.Thiswork was supportedbyCoordenac¸ãode

Aperfeic¸oamentodePessoaldeNívelSuperior(CAPES),

Con-selhoNacionaldeDesenvolvimentoCientíﬁcoeTecnológico

(CNPq) and Fundac¸ão Carlos Chagas Filho de Amparo à

PesquisadoEstadodoRiodeJaneiro(FAPERJ).

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